CN102918061A - Antibodies against human CSF-1R and uses thereof - Google Patents

Abstract

The present invention relates to antibodies against human CSF-1R (CSF-1R antibodies), methods of their production, pharmaceutical compositions containing them, and uses thereof.

Description

Antibodies against human CSF-1R and uses thereof

field of invention

The present invention relates to an antibody against human CSF-1R (CSF-1R antibody), a production method thereof, a pharmaceutical composition containing the antibody, and uses thereof.

Background of the invention

CSF-1 receptor (CSF-1R; synonyms: M-CSF receptor; macrophage colony-stimulating factor 1 receptor, EC 2.7.10.1, Fms proto-oncogene, c-fms, Swiss Prot P07333, CD115) since 1986 has been known since years (Coussens L. et al., Nature 320(1986) 277-280). CSF-IR is a growth factor and is encoded by the c-fms proto-oncogene (for review see eg Roth, P. and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-67).

CSF-1R is a receptor for M-CSF (macrophage colony-stimulating factor, also known as CSF-1), and mediates the biological effects of this cytokine (Sherr, C.J. et al., Cell 41(1985) 665-676 ). The cloning of the colony-stimulating factor-1 receptor (also known as c-fms) was first described by Roussel, M.F. et al., Nature 325 (1987) 549-552. In this publication, it was shown that CSF-1R has a transforming potential that is dependent on changes in the C-terminal tail of the protein, including loss of phosphorylation of the inhibitory tyrosine 969, which binds Cbl and thereby regulates receptor downregulation ( Lee, P.S. et al., Embo J. 18(1999) 3616-3628).

CSF-1R is a single-chain, transmembrane receptor tyrosine kinase (RTK) and member of the immunoglobulin (Ig) motif-containing RTK family characterized by repeated Ig domains in the extracellular portion of the receptor members. Intracellular protein tyrosine kinase domains are interrupted by unique insertion domains also present in receptors including platelet-derived growth factor receptor (PDGFR), stem cell growth factor receptor (c-Kit), and fins-like cytokine receptors. among other related RTK class III family members of the FLT3. Despite the structural homology between this family of growth factor receptors, they have distinct tissue-specific functions. CSF-1R is mainly expressed on cells of the monocyte lineage and in the female reproductive tract and placenta. In addition, Langerhans cells, a subset of smooth muscle cells already in the skin (Inaba, T. et al., J. Biol. Chem. 267 (1992) 5693-5699), B cells (Baker, A.H. et al., Expression of CSF-1R has been reported in Oncogene 8 (1993) 371-378) and in microglia (Sawada, M. et al., Brain Res. 509 (1990) 119-124).

The major biological effects of CSF-1R signaling are the differentiation, proliferation, migration, and survival of hematopoietic precursor cells to the macrophage lineage, including osteoclasts. Activation of CSF-1R is mediated by its ligand M-CSF. M-CSF binding to CSF-IR induces homodimer formation and kinase activation by tyrosine phosphorylation (Stanley, E.R. et al., Mol. Reprod. Dev. 46 (1997) 4-10). Additional signaling is mediated by the p85 subunit of PI3K and Grb2 linking the PI3K/AKT and Ras/MAPK pathways, respectively. These two important signaling pathways regulate proliferation, survival and apoptosis. Other signaling molecules that bind the phosphorylated intracellular domain of CSF-1R include STAT1, STAT3, PLCy, and Cbl (Bourette, R.P. and Rohrschneider, L.R., Growth Factors 17 (2000) 155-166).

CSF-1R signaling has physiological roles in the immune response, in bone remodeling and in the reproductive system. Knockout animals of M-CSF-1 (Pollard, J.W., Mol. Reprod. Dev. 46 (1997) 54-61) or CSF-1R (Dai, X.M. et al., Blood 99 (2002) 111-120) have been shown Osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes consistent with CSF-IR roles in the corresponding cell types.

Sherr, C.J. et al., Blood 73 (1989) 1786-1793 describe some antibodies against CSF-1R involved in the inhibition of CSF-1 activity (see Sherr, C.J. et al., Blood 73 (1989) 1786-1793). Ashum, R.A. et al., Blood 73 (1989) 827-837 relates to CSF-1R antibodies. Lenda, D.M. et al., Journal of immunology 170 (2003) 3254-3262 implicates reduced macrophage recruitment, proliferation, and activation in CSF-1 deficient mice leading to reduced tubular apoptosis during renal inflammation. Kitaura, H. et al., Journal of dental research 87 (2008) 396-400 mention an anti-CSF-1 antibody that inhibits orthodontic tooth movement. WO 2001/030381 mentions CSF-1 activity inhibitors including antisense nucleotides and antibodies in the context of disclosing only CSF-1 antisense nucleotides. WO 2004/045532 relates to metastasis and bone loss prevention and treatment of metastatic cancer by M-CSF antagonists (only anti-CSF-1 antibodies are disclosed as antagonists). WO 2005/046657 relates to the treatment of inflammatory bowel disease by anti-CSF-1 antibodies. US2002/0141994 relates to inhibitors of colony stimulating factor. WO 2006/096489 relates to the treatment of rheumatoid arthritis by anti-CSF-1 antibodies.

WO 2009/026303 and WO 2009/112245 relate to anti-CSF-1R antibodies.

Summary of the invention

The present invention includes an antibody binding to human CSF-1R, which is characterized in that it binds to the same epitope as the deposited antibody DSMACC2921.

In one embodiment, the antibody is characterized in comprising the CDR3 region of SEQ ID NO: 1 as the heavy chain variable domain CDR3 region.

In one embodiment, the antibody is characterized by:

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or

b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

In one embodiment, the antibody is characterized in comprising:

a) the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 7, and the amino acid sequence of the light chain variable domain is SEQ ID NO: 8; or

b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

In one embodiment, said antibody binds human CSF-1R and is characterized in that the above-mentioned amino acid sequences and amino acid sequence fragments are of human IgG1 subclass or of human IgG4 subclass.

Yet another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention.

The invention further includes pharmaceutical compositions characterized by comprising an antibody binding to human CSF-1R characterized by the above-mentioned epitope binding properties or alternatively by the above-mentioned Amino acid sequences and amino acid sequence fragments.

The invention further comprises the use of an antibody characterized by comprising an antibody binding to human CSF-1R characterized by the above mentioned epitope binding properties or alternatively for the manufacture of a pharmaceutical composition Amino acid sequences and amino acid sequence fragments mentioned above.

The present invention further comprises the use of an antibody characterized by comprising an antibody binding to human CSF-1R characterized by the above-mentioned epitope binding for the treatment of a CSF-1R mediated disease Properties or alternatively the above mentioned amino acid sequences and amino acid sequence fragments.

The invention further comprises the use of an antibody characterized by comprising an antibody binding to human CSF-1R characterized by the above-mentioned epitope binding properties or alternatively the above-mentioned Amino acid sequences and amino acid sequence fragments mentioned.

The invention further includes the use of an antibody characterized by comprising an antibody binding to human CSF-1R characterized by the above-mentioned epitope binding properties or alternatively by the above-mentioned Amino acid sequences and amino acid sequence fragments mentioned herein.

The invention further includes the use of an antibody characterized by comprising an antibody binding to human CSF-1R characterized by the above mentioned epitope binding properties or alternatively for the prevention or treatment of metastasis Amino acid sequences and amino acid sequence fragments mentioned above.

The invention further includes the use of an antibody characterized by comprising an antibody binding to human CSF-1R characterized by the above-mentioned epitope binding properties or alternatively, for the treatment of an inflammatory disease Amino acid sequences and amino acid sequence fragments mentioned above.

One aspect of the invention is an antibody that binds human CSF-1R, characterized in comprising the CDR3 region of SEQ ID NO: 1 as the heavy chain variable domain CDR3 region.

Another aspect of the invention is an antibody that binds to human CSF-1R, characterized in that

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or

b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

In one embodiment, the antibody is characterized in comprising

a) the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 7, and the amino acid sequence of the light chain variable domain is SEQ ID NO: 8; or

b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

In one aspect of the invention, an antibody according to the invention binds human CSF-1R with an affinity of at least 10 ⁻⁸ mol/l to 10 ⁻¹² mol/l.

In one aspect of the invention, the antibody according to the invention is a humanized antibody.

Yet another embodiment of the invention is a nucleic acid encoding a heavy chain variable domain and/or a light chain variable domain of an antibody according to the invention. Preferably, said nucleic acid encodes a heavy chain of an antibody that binds to human CSF-1R, characterized in that it comprises the CDR3 region of SEQ ID NO: 1 as the heavy chain CDR3 region.

Yet another embodiment of the invention is a nucleic acid encoding an antibody according to the invention, said antibody being characterized in that:

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or

b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

The invention further provides expression vectors containing nucleic acids according to the invention, capable of expressing said nucleic acids in prokaryotic or eukaryotic host cells, and host cells containing such vectors for the recombinant production of such antibodies.

The invention further includes prokaryotic or eukaryotic host cells comprising a vector according to the invention.

The invention further comprises a method for producing a recombinant humanized antibody according to the invention, characterized in that the nucleic acid according to the invention is expressed in a prokaryotic or eukaryotic host cell and recovered from said cells or cell culture supernatant the antibody. The invention further includes antibodies obtainable by such recombinant methods.

Antibodies according to the invention show benefit for patients in need of CSF-1R targeted therapy. The antibodies according to the invention have novel and inventive properties which are of benefit to patients suffering from neoplastic diseases, especially cancer.

The invention further provides a method for treating a patient with cancer comprising administering to a patient diagnosed with such disease (and therefore in need of such therapy) an effective amount of an antibody that binds human CSF-1R according to the invention. Preferably, the antibody is administered in a pharmaceutical composition.

Yet another embodiment of the invention is a method for the treatment of a patient suffering from cancer, characterized in that the antibody according to the invention is administered to the patient.

The invention further comprises the use of an antibody according to the invention for the treatment of a patient suffering from cancer and for the manufacture of a pharmaceutical composition according to the invention. Additionally, the present invention includes methods for the manufacture of pharmaceutical compositions according to the present invention.

The invention further comprises pharmaceutical compositions comprising an antibody according to the invention, optionally and buffers and/or adjuvants useful for formulating the antibody for pharmaceutical purposes.

The invention further provides a pharmaceutical composition comprising an antibody according to the invention in a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical compositions may be included in an article of manufacture or a kit.

Brief description of the drawings

Figure 1 : Growth inhibition of BeWo tumor cells in 3D culture under treatment with different anti-CSF-1R monoclonal antibodies at a concentration of 10 μg/ml.

X-axis: mean relative light units (RLU) of viability corresponding to the ATP content of the cells (CellTiterGlo assay).

Y axis: Test probes: minimal medium (0.5%FBS), mouse IgG1 (mIgG1, 10μg/ml), mouse IgG2a (mIgG2a 10μg/ml), CSF-1 only, <CSF-1R>7G5.3B6 , and SC-02, clone 2-4A5.

The highest inhibition of CSF-1 induced growth was observed with anti-CSF-1R antibodies according to the invention.

Detailed description of the invention

I. Definition

The term "antibody" encompasses various forms of antibodies, including but not limited to whole antibodies, antibody fragments, humanized antibodies, chimeric antibodies, T-cell epitope-depleted antibodies, and other genetically engineered antibodies, as long as they are in accordance with the present invention characteristic properties are preserved.

An "antibody fragment" comprises a portion of a full-length antibody, preferably, its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. For example, scFv antibodies are described in Huston, JS, Methods in Enzymol. 203 (1991) 46-88. In addition, antibody fragments comprise the characteristic of having a CSF-1R-binding _VH domain or a CSF-1R-binding _VL domain (i.e., capable of assembling together with a _VL or _VH domain into a functional antigen-binding site, and thereby providing This characteristic) single-chain polypeptide.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules composed of a single amino acid.

The term "chimeric antibody" refers to a monoclonal antibody comprising at least a portion of a mouse-derived variable region, ie, a binding region, and a constant region derived from a different source or species, usually produced by recombinant DNA techniques. Chimeric antibodies comprising mouse variable regions and human constant regions are particularly preferred. Such rat/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding rat immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of "chimeric antibodies" encompassed by the invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also known as "class switched antibodies". Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See eg Morrison, S.L. et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

As used within this application, the term "CDR-grafted variant" means a variant comprising complementarity determining regions (CDRs or hypervariable regions) from one source or species and CDRs from a different source or species, usually prepared by recombinant DNA techniques. Antibody variable domains in the framework regions (FR). CDR-grafted variants comprising murine CDRs and variable domains of human FRs are preferred.

As used within this application, the term "T-cell epitope-subtracted variant" means a variant modified to eliminate or reduce immunity by removing human T-cell epitopes (peptide sequences within variable domains that have the ability to bind MHC class II molecules). Antibody variable domains. By this method, the interactions between the amino acid side chains of the variable domains and specific binding pockets with the MHC class II binding groove are identified. The identified immunogenic regions were mutated to eliminate immunogenicity. Generally, such methods are described, for example, in WO 98/52976.

As used within this application, the term "humanized variant" means a complementarity determining region (CDR) of non-human origin, e.g. a non-human species, and a framework region (FR) of human origin reconstituted, and has Antibody variable domains that are further modified to also re-establish or improve the binding affinity and specificity of the original non-human variable domains. Such humanized variants are usually prepared by recombinant DNA techniques. Reconstitution of the affinity and specificity of the parental non-human variable domains is a crucial step, for which different approaches are currently used. In one approach, it is determined whether introducing mutations, so called back mutations, in non-human CDRs as well as in human FRs is beneficial. It can be done, for example, by sequence or homology analysis, by selecting a human framework (fixed framework approach; homology matching or best fit), by using consensus sequences, by selecting FRs from several different human mAbs, or by using The most common residues present in human mAbs were substituted for non-human residues on the three-dimensional surface (“resurfacing” or “veneering”) to identify suitable locations for such backmutations.

In addition, antibodies according to the present invention include such antibodies with "conservative sequence modifications", ie nucleotide and amino acid sequence modifications that do not affect or alter the above-mentioned characteristics of the antibodies according to the present invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine , isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine) amino acids. Thus, preferably, a predicted non-essential amino acid residue in a human anti-CSF-1R antibody may be replaced with another amino acid residue from the same side chain family.

Amino acid substitutions can be performed by mutagenesis based on molecular modeling as described by Riechmann, L. et al., Nature 332 (1988) 323-327 and Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033 describe.

As used herein, the term "CSF-1R" refers to human CSF-1R (SEQ ID No: 15). CSF-1R (synonyms: CSF-1 receptor; M-CSF receptor; macrophage colony-stimulating factor 1 receptor, EC 2.7.10.1, Fms proto-oncogene, c-fms, Swiss Prot P07333, CD115) since 1986 has been known since (Coussens L. et al., Nature 320(1986) 277-280). CSF-IR is a growth factor and is encoded by the c-fms proto-oncogene (for review see eg Roth, P. and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-67).

CSF-1R is a receptor for M-CSF (macrophage colony-stimulating factor, also known as CSF-1), and mediates the biological effects of this cytokine (Sherr, C.J. et al., Cell 41(1985) 665-676 ). The cloning of the colony-stimulating factor-1 receptor (also known as c-fms) was first described by Roussel, M.F. et al., Nature 325 (1987) 549-552. In this publication, it was shown that CSF-1R has a transforming potential that is dependent on changes in the C-terminal tail of the protein, including loss of phosphorylation of the inhibitory tyrosine 969, which binds Cbl, and thereby regulates receptor downregulation (Lee, P.S. et al., Embo J. 18(1999) 3616-3628).

CSF-1R is a single-chain, transmembrane receptor tyrosine kinase (RTK) and member of the immunoglobulin (Ig) motif-containing RTK family characterized by repeated Ig domains in the extracellular portion of the receptor members. Intracellular protein tyrosine kinase domains are interrupted by unique insertion domains also present in receptors including platelet-derived growth factor receptor (PDGFR), stem cell growth factor receptor (c-Kit), and fins-like cytokine receptors. among other related RTK class III family members of the FLT3. Despite the structural homology between this family of growth factor receptors, they have distinct tissue-specific functions. CSF-1R is predominantly found on cells of the monocyte lineage and in the female reproductive tract and placenta. In addition, Langerhans cells, a subset of smooth muscle cells already in the skin (Inaba, T. et al., J. Biol. Chem. 267 (1992) 5693-5699), B cells (Baker, A.H. et al., Expression of CSF-1R has been reported in Oncogene 8 (1993) 371-378) and in microglia (Sawada, M. et al., Brain Res. 509 (1990) 119-124).

)测定结合亲和力（见实施例4）。As used herein, the terms "bind to human CSF-1R" or "anti-CSF-1R" are used interchangeably and refer to antibodies that specifically bind human CSF-1R antigens. The binding affinity is a KD value of 1.0x10 ^-8 mol/l or lower at 35°C, preferably a KD value of 1.0x10 ^-9 mol/l or lower at 35°C. Using standard binding assays such as surface plasmon resonance (Biacore

) to determine binding affinity (see Example 4).

The term "epitope" means a determinant of a protein capable of specifically binding an antibody. Epitopes usually consist of chemically active surface aggregations of molecules such as amino acids or sugar side chains, and usually epitopes have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the loss of binding to the former, but not to the latter, in the presence of denaturing solvents. Preferably, the antibodies according to the invention specifically bind native, not denatured CSF-1R.

As used herein, the term "binding to the same epitope as the deposited antibody DSM ACC2921" refers to the antibody of the present invention that binds to the same epitope as the antibody <CSF-1R>7G5.3B6 (deposit number DSM ACC2921) on CSF-1R. Antibody to CSF-1R. The epitope binding properties of the anti-CSF-1R antibodies of the invention can be determined using techniques known in the art. CSF- 1R antibody. This can be investigated by BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden), as for example in Example 5. In Example 5, the percentage (%) of the expected binding response of the CSF-1R antibody of the invention competing with the bound antibody <CSF-1R>7G5.3B6 (Accession No. DSMACC2921) was calculated by "100*relative response (approximately_ Stability_early)/rMax", where rMax is calculated by "Relative Response (Mass_Stability_Late)*Antibody Molecular Weight/Antigen Molecular Weight" as described in the Biacore assay epitope localizer. Minimal binding responses were also calculated from pairs of the same antibodies 1 and 2 (see Example 5). The maximum value obtained by it + 100%, preferably 50% is set as the threshold for significant competition and thus significant binding to the same epitope (see Example 5, for antibody <CSF-1R> 7G5.3B6, the calculated threshold is 3+3 =6, preferably 3+1.5=4.5). Thus, an antibody that binds to human CSF-1R characterized by "binding to the same epitope as <CSF-1R> 7G5.3B6 (Accession No. DSM ACC2921)" has a percentage of the expected binding response of less than 6, preferably 4.5 (% ) (% expected binding response of less than 6, preferably less than 4.5).

In one aspect, the antibody according to the invention competes with the deposited antibody DSM ACC2921 for binding to human CSF-1R. Such binding competition can be determined using techniques known in the art. CSF was measured by testing the ability of the antibody to inhibit the binding of antibody <CSF-1R>7G5.3B6 (Accession No. DSM ACC2921) to human CSF-1R by surface plasmon resonance (SPR) assay in an in vitro competitive binding inhibition assay at 25°C -1R antibody. This can be investigated by BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden), as for example in Example 5.

As used herein, "variable domain" (light chain variable domain (VL), heavy chain variable domain (VH)) means each of the pair of light and heavy chain domains that are directly involved in antibody binding to antigen. The variable light and heavy chain domains have the same general structure, and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation, while the CDRs can form loops connecting the β-sheet structures. The CDRs in each chain maintain their three-dimensional structure through the framework regions and, together with the CDRs from the other chain, form an antigen-binding site. The heavy and light chain CDR3 regions of antibodies play a particularly important role in the binding specificity/affinity of antibodies according to the invention and thus provide a further object of the invention.

The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from "complementarity determining regions" or "CDRs." "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding and defines antibody properties. CDR and FR regions are defined according to the standard of Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from "hypervariable loops" Sure.

As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

As used within this application, the term "amino acid" means the group of naturally occurring carboxy alpha-amino acids, including alanine (three-letter code: ala, one-letter code: A), arginine (arg, R) , asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine ( gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M ), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine ( tyr, Y), and valine (val, V).

"Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, antibodies are purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase determined by HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-CSF-1R antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or in different vectors, and present in a host Such nucleic acid molecules at one or more locations in a cell.

"Native antibody" refers to naturally occurring immunoglobulin molecules of varying structure. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains disulfide-bonded. From N to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also called variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of their constant domains, antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (λ).

The term "package insert" is used to refer to instructions commonly included in commercial packages of therapeutic products that contain information on the indications, usage, dosage, administration, combination therapies, contraindications and/or warnings concerning the use of such therapeutic products Information.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and when any conservative substitutions are not considered part of the sequence identity, The percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes of the present invention, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with user documentation, has been filed with the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source. The ALIGN2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In the case of comparing amino acid sequences using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or can be expressed as A given amino acid sequence A) having or comprising a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows:

Fraction X/Y times 100

where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with respect to B will not be equal to the % amino acid sequence identity of B with respect to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

II. Compositions and Methods

In one aspect, the invention is based in part on the same epitope as the deposited antibody DSM ACC2921. Antibodies of the invention are useful, for example, in the diagnosis or treatment of cancer, inflammatory diseases, or bone loss; or in the prevention or treatment of metastasis.

Exemplary anti-CSF-1R antibodies

In one aspect, the invention provides antibodies that bind human CSF-1R. In certain embodiments, the anti-CSF-1R antibody is characterized in that it binds to the same epitope as the deposited antibody DSMACC2921.

Another aspect of the invention is an antibody that binds to human CSF-1R, characterized in that:

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or

b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

Another aspect of the invention is an antibody that binds to human CSF-1R, characterized in that:

a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or

b) a CDR-grafted, humanized or T-cell epitope-depleted antibody variant of said antibody, and

Has one or more of the following properties (as determined in the assays described in Examples 2, 3, 4, 6, 7 and 8):

-An anti-CSF-1R antibody inhibits CSF-1 binding to CSF-1R with an IC50 of 25 ng/ml or lower, in one embodiment, with an IC50 of 20 ng/ml or lower;

-An anti-CSF-1R antibody inhibits CSF-1-induced CSF-1R phosphorylation with an IC50 of 100 ng/ml or less, in one embodiment, with an IC50 of 50 ng/ml or less (in recombinant cells);

-An anti-CSF-1R antibody inhibits the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID No: 15) by 80% or more (compared to no antibody), preferably 90% or more;

- the anti-CSF-1R antibody inhibits the growth of BeWo tumor cells (ATCC CCL-98) by 80% or more (at an antibody concentration of 10 μg/ml; and compared with no antibody), preferably 90% or more;

- the anti-CSF-1R antibody inhibits macrophage differentiation (in one embodiment, the anti-CSF-1R antibody inhibits monocyte survival with an IC50 of 1.5 nM or less, preferably with an IC50 of 1.0 nM or less); or

- The anti-CSF-1R antibody binds to human CSF-1R at 35°C with a binding affinity of KD=1.0×10 ⁻⁹ mol/l or lower.

In another aspect, the anti-CSF-1R antibody according to the present invention comprises in the heavy chain variable domain (VH) sequence a) an amino acid sequence identical to SEQ ID NO: 3, or comprises 1 relative to SEQ ID NO: 3 , CDR1H with 2, or 3 amino acid residue substitutions, b) having the same amino acid sequence as SEQ ID NO: 2, or CDR2H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 2 and c) have the same amino acid sequence as SEQ ID NO: 1, or CDR3H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 1.

In certain embodiments, comprising a) having the same amino acid sequence as SEQ ID NO:3, or CDR1H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:3, b) having the same amino acid sequence as SEQ ID NO:3 ID NO:2 the same amino acid sequence, or CDR2H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:2, and c) having the same amino acid sequence as SEQ ID NO:1, or relative to The heavy chain variable domain (VH) sequence of SEQ ID NO: 1 comprising 1, 2, or 3 amino acid residue substitutions of CDR3H contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but includes all Anti-CSF-1R antibodies of the above sequence retain the ability to bind CSF-1R.

In another aspect, the anti-CSF-1R antibody according to the present invention comprises in the light chain variable domain (VL) sequence a) an amino acid sequence identical to SEQ ID NO: 6, or comprises 1 relative to SEQ ID NO: 6 , CDR1L with 2, or 3 amino acid residue substitutions, b) having the same amino acid sequence as SEQ ID NO: 5, or CDR2L comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 5, and c) a CDR3L having the same amino acid sequence as SEQ ID NO:4, or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:4.

In certain embodiments, comprising a) a CDR1L having the same amino acid sequence as SEQ ID NO:6, or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:6, b) having the same amino acid sequence as SEQ ID NO:6 ID NO:5 the same amino acid sequence, or CDR2L comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:5, and c) having the same amino acid sequence as SEQ ID NO:4, or relative to The light chain variable domain (VL) sequence of the CDR3L of SEQ ID NO:4 comprising 1, 2, or 3 amino acid residue substitutions contains substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but includes all Anti-CSF-1R antibodies of the above sequence retain the ability to bind CSF-1R.

In another aspect, the anti-CSF-1R antibody according to the present invention

- comprising in the heavy chain variable domain (VH) sequence a) a CDR1H having the same amino acid sequence as SEQ ID NO: 3, or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 3, b) has the same amino acid sequence as SEQ ID NO:2, or a CDR2H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:2, and c) has the same amino acid sequence as SEQ ID NO:1 sequence, or CDR3H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 1, and comprising d) the same amino acid as SEQ ID NO: 6 in the light chain variable domain (VL) sequence sequence, or CDR1L comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:6, e) having the same amino acid sequence as SEQ ID NO:5, or comprising 1, 2, or 3 amino acid residues relative to SEQ ID NO:5 2, or CDR2L with 3 amino acid residue substitutions, and f) having the same amino acid sequence as SEQ ID NO: 4, or CDR3L comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 4.

In another aspect, the anti-CSF-1R antibody according to the present invention

- comprising in the heavy chain variable domain (VH) sequence a) a CDR1H having the same amino acid sequence as SEQ ID NO: 3, or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 3, b) has the same amino acid sequence as SEQ ID NO:2, or a CDR2H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:2, and c) has the same amino acid sequence as SEQ ID NO:1 sequence, or CDR3H comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 1, and comprising d) the same amino acid as SEQ ID NO: 6 in the light chain variable domain (VL) sequence sequence, or CDR1L comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO:6, e) having the same amino acid sequence as SEQ ID NO:5, or comprising 1, 2, or 3 amino acid residues relative to SEQ ID NO:5 2, or CDR2L with 3 amino acid residue substitutions, and f) a CDR3L having the same amino acid sequence as SEQ ID NO: 4, or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 4; and

Anti-CSF-1R antibodies have one or more of the following properties (as determined in the assays described in Examples 2, 3, 4, 6, 7 and 8):

-An anti-CSF-1R antibody inhibits CSF-1 binding to CSF-1R with an IC50 of 25 ng/ml or lower, in one embodiment, with an IC50 of 20 ng/ml or lower;

-An anti-CSF-1R antibody inhibits CSF-1-induced CSF-1R phosphorylation with an IC50 of 100 ng/ml or less, in one embodiment, with an IC50 of 50 ng/ml or less (in recombinant cells);

-An anti-CSF-1R antibody inhibits the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID No: 15) by 80% or more (compared to no antibody), preferably 90% or more;

- the anti-CSF-1R antibody inhibits the growth of BeWo tumor cells (ATCC CCL-98) by 80% or more (at an antibody concentration of 10 μg/ml; and compared with no antibody), preferably 90% or more;

- the anti-CSF-1R antibody inhibits macrophage differentiation (in one embodiment, the anti-CSF-1R antibody inhibits monocyte survival with an IC50 of 1.5 nM or less, preferably with an IC50 of 1.0 nM or less); or

- The anti-CSF-1R antibody binds to human CSF-1R at 35°C with a binding affinity of KD=1.0×10 ⁻⁹ mol/l or lower.

Recombinant methods and compositions

Preferably, the antibodies according to the invention are produced by recombinant means. Such methods are generally known in the art and include expression of the protein in prokaryotic and eukaryotic cells followed by isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For protein expression, nucleic acids encoding the light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and from cells (from supernatant or in cell lysates) after) to recover the antibody.

Recombinant production of antibodies is well known in the prior art and is described, for example, in the review articles Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. -282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.

Antibodies may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Purification is performed by standard techniques, including base/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques known in the art to eliminate other cellular components or other contaminants, such as other cells nucleic acid or protein. See Ausubel, F. et al., eds. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NSO cells is described eg by Barnes, L.M. et al., Cytotechnology 32 (2000) 109-123; Barnes, L.M. et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described eg by Durocher, Y. et al., Nucl. Acids. Res. 30 (2002) E9. The cloning of variable domain is by Orlandi, R. etc., Proc.Natl.Acad.Sci.USA 86 (1989) 3833-3837; Carter, P. etc., Proc.Natl.Acad.Sci.USA 89 (1992) 4285- 4289; described by Norderhaug, L. et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., Christensen, K., in Cytotechnology 30 (1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199 describe.

Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide if it expresses a preprotein involved in the secretion of the polypeptide; it is operably linked to a coding sequence if a promoter or enhancer affects the transcription of the sequence; or A ribosome binding site is operably linked to a coding sequence if it is positioned such that it facilitates translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If no such sites exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

Monoclonal antibodies are suitably isolated from culture broth by conventional immunoglobulin purification procedures such as, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using routine procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector that is then transfected into a host cell that does not otherwise produce immunoglobulin protein, such as HEK 293 cells, CHO cells, or myeloma cells, to obtain recombinant monoclonal antibodies Synthesis in host cells.

Nucleic acid molecules encoding amino acid sequence variants of anti-CSF-1R antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally-occurring amino acid sequence variants) or by oligonucleotide processing of variant or non-variant versions of humanized anti-CSF-1R antibodies prepared earlier. acid-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

The heavy and light chain variable domains according to the invention are combined with sequences for promoter, translation initiation, constant region, 3' untranslated region, polyadenylation, and transcription termination to form an expression vector construct. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a single host expressing both chains cell.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It should also be understood that all progeny may not be exactly identical in DNA content, due to deliberate or unintentional mutations. Variant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

The "Fc portion" of an antibody is not directly involved in binding of the antibody to antigen, but exhibits various effector functions. "Fc portion of an antibody" is a term well known to the skilled artisan and is defined based on cleavage of the antibody by papain. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgAl, and IgA2. According to the heavy-chain constant regions, the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity), which are based on complement activation, Clq binding and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Binding to C1q is caused by a defined binding site in the Fc portion, although the effect of the antibody on the complement system is dependent on certain conditions. Such binding sites are known in the prior art and are described, for example, in Boackle, R.J. et al., Nature 282 (1979) 742-743, Lukas, T.J. et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse , R. and Cebra, J.J., Mol. Immunol.16 (1979) 907-917, Burton, D.R. et al., Nature 288 (1980) 338-344, Thommesen, J.E. et al., Mol. Immunol.37 (2000) 995-1004 , Idusogie, E.E. et al., J.Immunol.164(2000) 4178-4184, Hezareh, M. et al., J.Virology 75(2001) 12161-12168, Morgan, A. et al., Immunology 86(1995) 319-324, EP 0307434. Such binding sites are eg L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat, E.A., see below). Antibodies of the subclasses IgG1, IgG2, and IgG3 usually show complement activation and C1q and C3 binding, whereas IgG4 does not activate the complement system and does not bind C1q and C3.

In one embodiment, an antibody according to the invention comprises an Fc part derived from human origin, and preferably all other parts of the human constant region. As used herein, the term "Fc portion derived from human origin" means an Fc portion as the Fc portion of a human antibody of the IgG1, IgG2, IgG3 or IgG4 subclass, preferably, from the human IgG1 subclass Fc part from human IgG1 subclass (preferably with mutations in L234A+L235A), Fc part from human IgG4 subclass or mutant Fc part from human IgG4 subclass (preferably with mutations in S228P mutation). Generally preferred are SEQ ID NO: 11 (human IgG1 subclass), SEQ ID NO: 12 (human IgG1 subclass with mutations L234A and L235A), SEQ ID NO: 13 (human IgG4 subclass), or SEQ ID NO Human heavy chain constant region of :14 (human IgG4 subclass with mutation S228P).

In one embodiment, the antibody according to the invention is characterized in that the invariant chain is of human origin. Such invariant chains are well known in the prior art and are eg described by Kabat, E.A. (see eg Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, one useful human heavy chain constant region comprises the amino acid sequence of SEQ ID NO:9. For example, a useful human light chain constant region comprises the amino acid sequence of a kappa light chain constant region of SEQ ID NO: 10. It is further preferred that the antibody is of mouse origin and comprises the antibody variable sequence frame of a mouse antibody according to Kabat.

Immunoconjugate

The present invention also provides compounds comprising and one or more cytotoxic agents, such as chemotherapeutics or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) ), or a radioisotope-conjugated immunoconjugate of the anti-CSF-1R antibody herein.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Patent No. 5,208,020 , 5,416,064 and European Patent EP 0 425 235B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); Calicheamicin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342); and (1Lo93 , Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al., CurrentMed.Chem.13:477-523 (2006 ); Jeffrey et al, Bioorganic & Med.Chem.Letters 16:358-362 (2006); Torgov et al, Bioconj.Chem.16:717-721 (2005); Nagy et al, Proc.Natl.Acad.Sci.USA 97:829- 834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin, or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin , exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α - Inhibition of sarcin, Aleutites fordii toxin, dianthin toxin, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia Curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin , phenomycin, enomycin and trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the generation of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and radioactive isotopes of Lu. When radioconjugates are used for detection, it can contain radioactive atoms such as tc99m or I123 for scintillation studies, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) substances such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents can be used to generate conjugates of antibodies and cytotoxic agents, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl -4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imidate (such as dimethyl adipimidate hydrochloride esters), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azides (such as bis(p-azidobenzoyl)hexamethylenediamine), double Nitrogen derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-reactive fluorine compounds (such as 1,5-difluoro-2, 4-Dinitrobenzene) bifunctional derivatives. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res 52:127-131 (1992); U.S. Patent No. 5,208,020 ).

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl -(4-vinylsulfone)benzoate), which are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

Therapeutic methods and compositions

The invention includes a method for treating a patient in need thereof, characterized in that a therapeutically effective amount of an antibody according to the invention is administered to the patient.

The invention includes the use of an antibody according to the invention for therapy.

A preferred embodiment of the present invention is the CSF-1R antibody of the present invention for use in the treatment of "CSF-1R-mediated diseases" or for the manufacture of "CSF-1R-mediated diseases" (which can be described as follows ) of the CSF-1R antibody of the invention as a drug.

There are 3 unique mechanisms by which CSF-IR signaling may be involved in tumor growth and metastasis. The first is that the expression of CSF ligands and receptors has been found in tumor cells originating in the female reproductive system (breast, ovary, endometrium, cervix) (Scholl, S.M. et al., J. Natl. Cancer Inst. 86( 1994) 120-126; Kacinski, B.M., Mol.Reprod.Dev.46(1997) 71-74; Ngan, H.Y. et al., Eur.J.Cancer 35(1999) 1546-1550; Kirma, N. et al., Cancer Res 67 (2007) 1918-1926), and expression has been linked to breast cancer xenograft growth and poor prognosis in breast cancer patients. Two point mutations were seen in CSF-1R in approximately 10-20% of patients with acute myeloid leukemia, chronic myelogenous leukemia and myelodysplasia tested in one study, and one of the mutations was found to disrupt receptor turnover (Ridge, S.A. et al., Proc. Natl. Acad. Sci USA 87 (1990) 1377-1380). However, the occurrence of the mutation could not be confirmed in later studies (Abu-Duhier, F.M. et al., Br. J. Haematol. 120 (2003) 464-470). Also in hepatocellular carcinoma (Yang, D.H. et al., Hepatobiliary Pancreat. Dis. Int. 3 (2004) 86-89) and idiopathic myelofibrosis (Abu-Duhier, F.M. et al., Br.J.Haematol.120 (2003 )464-470) mutations were found in some cases.

Pigmented villonodular synovitis (PVNS) and tenosynovial giant cell tumor (TGCT) can occur as a result of a translocation that fuses the M-CSF gene to the collagen gene COL6A3, leading to M-CSF overexpression (West, R.B. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 690-695). A landscape effect is proposed to account for the resulting tumor mass consisting of monocytes attracted to M-CSF expressing cells. TGCTs are small tumors that can be removed relatively easily from where they usually arise. PVNS is more aggressive because it can recur in large joints and is not as easy to control surgically.

The second mechanism is based on blocking signaling via M-CSF/CSF-IR at metastatic sites in bone, which induces osteoclastogenesis, bone resorption and osteolytic bone damage. Breast cancer, multiple myeloma, and lung cancer are examples of cancers that have been found to metastasize to bone and cause osteolytic bone disease, leading to skeletal complications. M-CSF released by tumor cells and stroma cooperates with the receptor activator of nuclear factor kappa-B ligand RANKL to induce differentiation of hematopoietic myeloid mononuclear progenitors into mature osteoclasts. During this process, M-CSF acts as a permissive factor by imparting a survival signal to osteoclasts (Tanaka, S. et al., J. Clin. Invest. 91 (1993) 257-263). Inhibition of CSF-1R activity during osteoclast differentiation and maturation with anti-CSF-1R antibodies has the potential to prevent the unbalanced activity of osteoclasts responsible for bone-related events involved in osteolytic and metastatic disease. Whereas breast, lung cancer, and multiple myeloma often result in osteolytic lesions, bone metastases in prostate cancer initially have an osteoblastic appearance in which elevated bone-forming activity results in "woven bone" that differs from the typical lamellae of normal bone. The structure is different. During disease progression, bone lesions exhibit a marked osteolytic component and high serum levels of bone resorption, and suggest that antiresorptive therapy may be useful. Bisphosphonates have been shown to inhibit the formation of osteolytic lesions and reduce the number of bone-related events only in men with hormone-refractory metastatic prostate cancer, but at this point, their effect on osteoblast damage The effect of is controversial, and bisphosphonates have not been beneficial in preventing bone metastases or hormone-responsive prostate cancer to date. The effect of antiresorptive agents in mixed osteolytic/osteogenic prostate cancer is still under investigation in the clinic (Choueiri, M.B. et al., Cancer Metastasis Rev. 25 (2006) 601-609; Vessella, R.L. and Corey, E. ., Clin. Cancer Res. 12 (20Pt 2) (2006) 6285s-6290s).

The third mechanism is based on recent observations that tumor-associated macrophages (TAMs) present in solid tumors of breast, prostate, ovary, and cervix are associated with poor prognosis (Bingle, L. et al., J. Pathol .196 (2002) 254-265; Pollard, J.W., Nat. Rev. Cancer 4 (2004) 71-78). Macrophages are recruited to tumors by M-CSF and other chemokines. Macrophages can then contribute to tumor progression via secretion of angiogenic factors, proteases, and other growth factors and cytokines, and can be blocked by inhibiting CSF-1R signaling. Recently, Zins, K. et al. (Zins, K. et al., CancerRes. 67 (2007) 1038-1045) showed that expression of siRNA for tumor necrosis factor alpha (TNFα), M-CSF, or a combination of both Intratumoral injection of siRNA reduces tumor growth in mouse xenograft models by 34%-50%. siRNA targeting TNFα secreted by human SW620 cells decreased M-CSF levels in mice and resulted in decreased macrophages in tumors. In addition, treatment of MCF7 tumor xenografts with antigen-binding fragments directed against M-CSF did result in 40% tumor growth inhibition, reversed resistance to chemotherapeutic agents, and improved survival of mice when given in combination with chemotherapeutic agents (Paulus, P. et al., Cancer Res. 66(2006) 4349-4356).

TAMs are the only example of an emerging link between chronic inflammation and cancer. There is additional evidence for a link between inflammation and cancer, as many chronic diseases are associated with increased cancer risk, cancer arises at sites of chronic inflammation, and chemical mediators of inflammation are found in many cancers; cells or chemicals that delete inflammation The mediator inhibits the formation of experimental cancers, and long-term use of the anti-inflammatory reduces the risk of some cancers. Those with H. pylori-induced gastritis (for stomach cancer), schistosomiasis (for bladder cancer), HHVX (for Kaposi sarcoma), endometriosis (for ovarian cancer ) and prostatitis (for prostate cancer), there are many inflammatory conditions associated with cancer (Balkwill, F. et al., Cancer Cell 7 (2005) 211-217). Macrophages are key cells in chronic inflammation and respond differentially to their microenvironment. There are two classes of macrophages that are considered extreme on a continuum of functional states: M1 macrophages are involved in type 1 responses. These reactions involve the activation and subsequent killing of pathogenic microorganisms by microbial products, which generate reactive oxygen intermediates. At the other end of the extreme are M2 macrophages involved in type 2 responses that promote cell proliferation, regulate inflammation and adaptive immunity, but also promote tissue remodeling, angiogenesis and repair (Mantovani, A. et al., Trends Immunol .25 (2004) 677-686). Chronic inflammation leading to the formation of established neoplasms is often associated with M2 macrophages. One of the key cytokines mediating inflammatory responses is TNFα, which, as its name suggests, stimulates anti-tumor immunity and hemorrhagic necrosis in high doses, and has recently been found to be expressed by tumor cells and acts as a tumor promoter ( Zins, K. et al., Cancer Res. 67 (2007) 1038-1045; Balkwill, F., Cancer Metastasis Rev. 25 (2006) 409-416). The specific roles of macrophages with respect to tumors still need to be better understood, including potential spatial and temporal dependencies of their function and relevance to specific tumor types.

Thus, one embodiment of the invention is a CSF-IR antibody of the invention for use in the treatment of cancer. As used herein, the term "cancer" may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye Tumor, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastric cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer , vulvar cancer, Hodgkin's disease, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral carcinoma, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary cancer, central nervous system (CNS) neoplasm, spinal axis tumor, brainstem glioma, glioma multiforme Glioblastoma multiforme, astrocytoma, schwanoma, ependymona, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphoma Leukemia, including refractory forms of any of the above cancers, or a combination of one or more of the above cancers. Preferably, such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. Preferably, such cancers are further characterized by CSF-1 or CSF-1R expression or overexpression. Yet another embodiment of the invention is the CSF-IR antibody of the invention for use in the simultaneous treatment of primary tumors and new metastases.

Thus, another embodiment of the present invention is in the treatment of periodontitis, histiocytosis X, osteoporosis, Paget's disease of bone (PDB), bone loss due to cancer therapy, prosthetic Peripheral osteolysis, glucocorticoid-induced osteoporosis, rheumatoid arthritis, psiratic arthritis, osteoarthritis, inflammatory arthritis, and inflammation CSF-1R antibodies of the invention.

Rabello, D. et al., Biochem. Biophys. Res. Commun. 347 (2006) 791-796 have shown that a SNP in the CSF1 gene exhibits an association with aggressive periodontitis: a disease of tooth loss due to alveolar bone resorption. Positive association with periodontal inflammatory disease.

Histiocytosis X (also known as Langerhans cell histiocytosis, LCH) is a proliferative disorder of Langerhans dendritic cells that differentiate into osteoclasts in bony and extraskeletal LCH lesions. disease. Langerhans cells are derived from circulating monocytes. Elevated M-CSF levels measured in serum and lesions were found to correlate with disease severity (da Costa, C.E. et al., J. Exp. Med. 201 (2005) 687-693). The disease occurs mainly in the pediatric patient population and has to be treated with chemotherapy when the disease becomes systemic or recurrent.

The pathophysiology of osteoporosis is mediated by loss of bone-forming osteoblasts and increased osteoclast-dependent bone resorption. Cenci et al. have described supporting data showing that anti-M-CSF antibody injections protect bone mineral density and inhibit bone resorption in ovariectomized mice (Cenci, S. et al., J. Clin. Invest. 105 (2000) 1279 -1287). Recently, a potential link between postmenopausal bone loss due to estrogen deficiency was identified and the presence of TNFα-producing T cells was found to affect bone metabolism (Roggia, C. et al., Minerva Med. 95 (2004) 125-132) . One possible mechanism could be that TNFα induces M-CSF in vivo. The essential role of M-CSF in TNF-α-induced osteoclastogenesis was confirmed by the effect of antibodies against M-CSF in blocking TNFα-induced osteolysis in mice, and thus generation of inflammatory arthritic CSF Inhibitors of potential targets of -1R signaling (Kitaura, H. et al., J. Clin. Invest. 115 (2005) 3418-3427).

Paget's disease of bone (PDB) is the second most common disorder of bone metabolism after osteoporosis, in which focal abnormalities of increased bone turnover lead to complications such as bone pain, deformity, pathological fractures and deafness. Mutations in four genes that regulate normal osteoclast function and predispose individuals to PDB and related disorders have been identified: TNFRSF11A (which encodes the receptor activator of nuclear factor (NF)κB (RANK), which a critical regulator of osteoclast function), inactivating mutations in TNFRSF11B encoding osteoprotegerin (a decoy receptor for RANK ligand), Sequestosome1 gene (SQSTM1) (which encodes important scaffold proteins) and mutations in the valoropeptide-containing protein (VCP) gene. This gene encodes a VCP that has a role in targeting NFKB inhibitors for degradation by the proteasome (Daroszewska, A. and Ralston, S.H., Nat. Clin. Pract. Rheumatol. 2 (2006) 270-277). Targeted CSF-1R inhibitors offer an opportunity to indirectly block deregulation of RANKL signaling and add additional therapeutic options to currently used bisphosphonates.

Cancer therapy-induced bone loss, especially in breast and prostate cancer patients, is an additional indication where targeted CSF-1R inhibitors can prevent bone loss (Lester, J.E. et al., Br.J.Cancer94 (2006 )30-35). As the prognosis of early breast cancer improves, the long-term consequences of adjuvant therapy become more important because some therapies, including chemotherapy, radiation, aromatase inhibitors, and ovariectomy affect bone metabolism by reducing bone mineral density, leading to osteoporosis and associated increased risk of fractures (Lester, J.E. et al., Br. J. Cancer 94 (2006) 30-35). The equivalent of adjuvant aromatase inhibitor therapy in breast cancer is androgen ablation therapy in prostate cancer, which results in loss of bone mineral density and significantly increases the risk of osteoporosis-related fractures (Stoch, S.A. et al., J. Clin. Endocrinol. Metab. 86 (2001) 2787-2791).

Targeted inhibition of CSF-1R signaling may also be beneficial in other indications when targeting cell types including osteoclasts and macrophages, such as therapy in response to joint replacement (due to rheumatoid arthritis ) specific complications. Implant failure due to periprosthetic bone loss and subsequent prosthetic laxity is a major complication of joint replacement surgery and requires repeat surgery with a high socioeconomic burden on the individual patient and the healthcare system. To date, there is no approved drug therapy for preventing or inhibiting periprosthetic osteolysis (Drees, P. et al., Nat. Clin. Pract. Rheumatol. 3 (2007) 165-171).

Glucocorticoid-induced osteoporosis (GIOP) is another indication in which CSF-1R inhibitors can prevent long-term glucose given for various conditions of chronic obstructive pulmonary disease, asthma and rheumatoid arthritis Bone loss following corticosteroid use (Guzman-Clark, J.R. et al., Arthritis Rheum. 57 (2007) 140-146; Feldstein, A.C. et al., Osteoporos. Int. 16 (2005) 2168-2174).

Rheumatoid arthritis, psoriatic arthritis, and inflammatory arthritis themselves are potential indications for CSF-1R signaling inhibitors because they consist of a macrophage component and varying degrees of bone destruction (Ritchlin, C.T. et al., J. Clin. Invest. 111 (2003) 821-831). Osteoarthritis and rheumatoid arthritis are inflammatory autoimmune diseases caused by the accumulation of macrophages in connective tissue and the infiltration of macrophages into synovial fluid, which is at least partially mediated by M-CSF. Campbell, I.K. et al., J.Leukoc.Biol.68 (2000) 144-150 show that human-articular tissue cells (chondrocytes, synovial fibroblasts) produce M-CSF in vitro and that it is present in rheumatoid arthritis in the synovial fluid of patients, suggesting that it contributes to the infiltration of macrophages and proliferation of synovial tissue associated with the pathogenesis of the disease. Inhibition of CSF-1R signaling has the potential to control the number of macrophages in joints and alleviate pain from the associated bone destruction. To minimize adverse effects and to further understand the impact of CSF-1R signaling in these indications, one approach is to specifically inhibit CSF-1R without targeting myriad other kinases, such as Raf kinases.

Recent literature reports link increased circulating M-CSF to poor prognosis and progression of atherosclerosis in chronic coronary artery disease (Saitoh, T. et al., J. Am. Coll. Cardiol. 35 (2000) 655-665 ; Ikonomidis, I. et al., Eur.Heart.J.26 (2005) pp. 1618-1624); M-CSF acts by helping to form foam cells that express CSF-1R and present initial plaques (with uptake of oxidized LDL Macrophages) affect the atherosclerotic process (Murayama, T. et al., Circulation 99 (1999) 1740-1746).

Expression and signaling of M-CSF and CSF-1R were found in activated microglia. Microglia, which are resident macrophages of the central nervous system, can be activated by a variety of insults, including infection and traumatic injury. M-CSF is thought to be a key regulator of inflammatory responses in the brain, and M-CSF levels are elevated in HIV-1, encephalitis, Alzheimer's disease (AD) and brain tumors. Microgliosis due to autocrine signaling of M-CSF/CSF-1R leads to induction of inflammatory cytokines and nitric oxide release, as demonstrated by, for example, using an experimental neuronal injury model (Hao , A.J. et al., Neuroscience 112 (2002) 889-900; Murphy, G.M., Jr. et al., J. Biol. Chem. 273 (1998) 20967-20971). Microglia with elevated CSF-1R expression were found to surround plaques in AD and in an amyloid precursor protein V717F transgenic mouse model of AD (Murphy, G.M., Jr. et al., Am. J. Pathol. 157 (2000) 895-904). On the other hand, op/op mice with fewer microglia in the brain resulted in fibril deposition of A-β and neuronal loss compared with normal controls, suggesting a role for microglia in op/op mice It does have a neuroprotective function in the development of AD deficiency (Kaku, M. et al., Brain Res. Brain Res. Protoc. 12 (2003) 104-108).

The expression and signaling of M-CSF and CSF-IR are associated with inflammatory bowel disease (IBD) (WO2005/046657). The term "inflammatory bowel disease" refers to severe, chronic intestinal disorders characterized by chronic inflammation at multiple sites in the gastrointestinal tract, and specifically includes ulcerative colitis (UC) and Crohn's disease.

The present invention includes antibodies for use in the treatment of cancer characterized by comprising antibodies binding to human CSF-1R characterized by the above mentioned epitope binding properties or alternatively by the above mentioned Amino acid sequences and amino acid sequence fragments.

The present invention includes antibodies for use in the treatment of bone loss characterized by comprising antibodies binding to human CSF-1R characterized by the above mentioned epitope binding properties or alternatively by the above mentioned And amino acid sequences and amino acid sequence fragments.

The present invention includes antibodies for the prevention or treatment of metastasis characterized by comprising antibodies binding to human CSF-1R characterized by the above mentioned epitope binding properties or alternatively by the above Amino acid sequences and amino acid sequence fragments mentioned.

The present invention includes antibodies for use in the treatment of inflammatory diseases characterized by comprising antibodies binding to human CSF-1R characterized by the above mentioned epitope binding properties or alternatively by the above Amino acid sequences and amino acid sequence fragments mentioned.

The present invention includes the use of an antibody for the treatment of cancer or alternatively for the manufacture of a medicament for the treatment of cancer, said antibody being characterized in that it comprises an antibody that binds to human CSF-1R, said antibody that binds to human CSF-1R is characterized by the above The epitope binding properties mentioned herein or alternatively the amino acid sequences and amino acid sequence fragments mentioned above.

The present invention includes the use of an antibody for the treatment of bone loss or alternatively for the manufacture of a medicament for the treatment of bone loss, said antibody being characterized by comprising an antibody that binds to human CSF-1R, said antibody that binds to human CSF-1R is characterized by In the above mentioned epitope binding properties or alternatively in the above mentioned amino acid sequences and amino acid sequence fragments.

The present invention includes the use of an antibody for preventing or treating metastasis or alternatively for the manufacture of a medicament for preventing or treating metastasis, said antibody being characterized in that it comprises an antibody that binds to human CSF-1R, said human CSF-1R that binds Antibodies are characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments.

The present invention includes the use of an antibody for the treatment of an inflammatory disease or alternatively for the manufacture of a medicament for the treatment of an inflammatory disease, said antibody being characterized in that it comprises an antibody that binds to human CSF-1R, said human CSF-1R that binds Antibodies are characterized by the above mentioned epitope binding properties or alternatively by the above mentioned amino acid sequences and amino acid sequence fragments.

In one embodiment, the antibody according to the invention inhibits CSF-1 binding to CSF-1R with an IC50 of 25 ng/ml or lower, preferably with an IC50 of 20 ng/ml or lower. The IC50 for inhibition of CSF-1 binding to CSF-1R can be determined as shown in Example 2.

In one embodiment, the antibody according to the invention inhibits CSF-1 induction with an IC50 of 100 ng/ml or less, preferably with an IC50 of 50 ng/ml or less, more preferably with an IC50 of 25 ng/ml or less CSF-1R phosphorylation (in NIH3T3-CSF-1R recombinant cells). The IC50 of CSF-1-induced phosphorylation of CSF-1R can be determined as shown in Example 3.

In one embodiment, the antibody according to the present invention inhibits the growth of recombinant NIH3T3 cells expressing human CSF-1R (SEQ ID No: 15) by 80% or more (compared to no antibody), preferably 90% or more many. % growth inhibition was determined as shown in Example 6, where % survival was measured. From % survival, % growth inhibition was calculated as follows: % growth inhibition=100-% survival. For example <CSF-1R>7G5.3B6 showed 100-0=100% growth inhibition of NIH3T3 cells expressing wt human CSF-1R.

In one embodiment, the antibody according to the invention stimulates the growth of recombinant NIH3T3 cells expressing human mutant CSF-1R L301SY969F (SEQ ID No: 16) by 5% or more (compared to no antibody), preferably 20 %Or more. % growth stimulation was determined as shown in Example 6, where % survival was measured. From % Survival, % Growth Stimulation was calculated as follows: % Growth Stimulation = -(100-% Survival). For example, <CSF-1R>7G5.3B6 showed -(100-0)=-(100-140)%=+40% growth stimulation of NIH3T3 cells expressing mutant human CSF-1R.

In one embodiment, the antibody according to the invention inhibits the growth of BeWo tumor cells (ATCC CCL-98) by 80% or more (antibody concentration is 10 μg/ml; and compared to no antibody), preferably 90% or More. % growth inhibition was determined as shown in Example 7. For example <CSF-1R>7G5.3B6 showed 101% growth inhibition of BeWo tumor cells.

In one embodiment, an antibody according to the invention inhibits macrophage differentiation. In one embodiment, the antibody according to the invention inhibits monocyte survival with an IC50 of 1.5 nM or lower, preferably with an IC50 of 1.0 nM or lower. Inhibition of monocyte survival was determined as shown in Example 8.

Yet another embodiment of the present invention is a method for producing antibodies against CSF-1R, characterized in that the nucleic acid encoding the heavy chain of a human IgG1 class antibody that binds to human CSF-1R according to the present invention (the modified nucleic acid ) and the nucleic acid sequence encoding the light chain of the antibody are inserted into an expression vector, the vector is inserted in a eukaryotic host cell, the encoded protein is expressed, and recovered from the host cell or supernatant.

pharmaceutical preparations

The term "pharmaceutical formulation" refers to a formulation which is in such a form as to allow the biological activity of the active ingredients contained therein to be effective and which contains no other substances which are unacceptably toxic to a subject to whom the formulation will be administered. components.

In another aspect, the invention provides compositions, eg, pharmaceutical compositions, comprising one or a panel of monoclonal antibodies or antigen-binding portions thereof of the invention formulated together with a pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion.

Compositions of the invention can be administered by a variety of methods known in the art. As the skilled artisan will appreciate, the route and/or mode of administration will vary with the desired result.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Besides water, the carrier can be, for example, an isotonic buffered saline solution.

Irrespective of the chosen route of administration, the compounds of the invention (which may be used in suitably hydrated form) and/or the pharmaceutical compositions of the invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve a particular patient's desired therapeutic response, composition, and mode of administration, without being toxic to the patient (effective amount) . The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, or its ester, salt or amide, the route of administration, time of administration, the rate of excretion of the particular compound employed. , other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated are factors well known in the medical art.

The present invention includes the use of an antibody according to the invention for the treatment of a patient suffering from cancer, especially colon, lung or pancreatic cancer.

The invention also includes methods for treating patients suffering from such diseases.

The present invention further provides methods for the manufacture of pharmaceutical compositions comprising an effective amount of an antibody according to the invention and a pharmaceutically acceptable carrier and the use of an antibody according to the invention for such methods.

The invention further provides the use of an effective amount of an antibody according to the invention for the manufacture of a medicament, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

The present invention also provides the use of an effective amount of an antibody according to the present invention for the manufacture of a medicament, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

products

In another aspect of the invention there is provided an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above. Articles of manufacture comprising containers and labels or package inserts on or associated with containers. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition effective to treat, prevent, and/or diagnose a condition, alone or in combination with another composition, and can have a sterile access opening (e.g., the container can be a tube with a stopper passable by a hypodermic needle vial or bag of intravenous solution). At least one active agent in the composition is an antibody of the invention. The label or package insert directs use of the composition to treat the condition of choice. Additionally, the article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises additional cells Toxic or otherwise therapeutic agents. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be appreciated that any of the foregoing preparations may comprise an immunoconjugate of the invention in place of or in addition to the anti-CSF-1R antibody.

The following examples and sequence listing are provided to aid in the understanding of the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made in the procedures set forth without departing from the spirit of the invention.

Antibody storage

cell line Accession number Preservation date <CSF-1R>7G5.3B6 DSM ACC2921 10.06.2008

sequence description

The following examples, sequence listing and figures are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made in the procedures set forth without departing from the spirit of the invention.

III. Example

Example 1

Generation of hybridoma cell lines producing anti-CSF-1R antibodies

Immunization protocol for NMRI mice

NMRI mice were immunized with the expression vector pDisplay ^™ (Invitrogen, USA) encoding the extracellular domain of huCSF-1R by using electroporation. Each mouse was immunized 4 times with 100 μg DNA. When serum titers against huCSF-1R were found to be sufficient, mice were additionally treated intravenously (iv) with 50 μg of the 1:1 mixture huCSF-1R ECD/huCSF-1R ECDhuFc chimera in 200 μl PBS 4 and 3 days before fusion. Strengthen once.

antigen-specific ELISA

Anti-CSF-1R titers in sera of immunized mice were determined by antigen-specific ELISA.

溶液(1mg/ml)（ABTS：2,2’-连氮基二(3-乙基苯并噻唑啉-6-磺酸)）显现测定法10分钟。于405nm测量吸光度。Capture 0.3 on Streptavidin plates (MaxiSorb; MicroCoat, DE, Cat. No. 11974998/MC1099) with 0.1 mg/ml biotinylated anti-Fcγ (Jackson ImmunoResearch., Cat. No. 109-066-098). μg/ml huCSF 1R-huFc chimera (soluble ectodomain) with addition of horseradish peroxidase (HRP)-conjugated F(ab ') 2 anti-mouse IgG (GE Healthcare, UK, catalog number NA9310V). Sera from all taps were diluted 1/40 in PBS/0.05% Tween20/0.5% BSA and serially diluted up to 1/1638400. Diluted sera were added to the wells. Serum before aspirating was used as a negative control. A dilution series of mouse anti-human CSF-IR mAb 3291 (R&D Systems, UK) from 500 ng/ml to 0,25 ng/ml was used as positive control. All components were incubated together for 1,5 hours. The wells were washed 6 times with PBST (PBS/0.2% Tween20) and washed with freshly prepared ABTS at RT

Solution (1 mg/ml) (ABTS: 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) developed the assay for 10 minutes. Absorbance was measured at 405 nm.

hybridoma generation

Mouse lymphocytes can be isolated and fused with mouse myeloma cell lines using standard PEG-based protocols to generate hybridomas. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of spleen-derived lymphocytes from immunized mice were fused with Ag8 non-secreting mouse myeloma cells P3X63Ag8.653 (ATCC, CRL-1580) with 50% PEG. Cells were distributed in approximately ¹⁰⁴ in flat-bottomed 96-well microtiter plates, followed by incubation in selective medium for approximately 2 weeks. Individual wells were then screened for human anti-CSF-IR monoclonal IgM and IgG antibodies by ELISA. Once extensive hybridoma growth has occurred, antibody-secreting hybridomas are redistributed, screened again, and if still positive for human IgG, anti-CSF-1R monoclonal antibodies can be subcloned by FACS. Stable subclones are then grown in vitro to produce antibodies in tissue culture for characterization.

Hybridoma culture

The resulting murine mAb hybridomas were treated in the presence of 2 mM L-Glutamine (GIBCO - Catalog No. 35050-038), 1 mM Sodium Pyruvate (GIBCO - Catalog No. 11140-035), 10% FCS (PAA - Catalog No. A15-649), 1x Pen Strep (Roche - Catalog No. 1074440), 1x Nutridoma CS (Roche - Catalog No. 1363743), 50 μM Mercaptoethanol (GIBCO - Catalog No. Cat. No. 31350-010) and 50 U/ml IL 6 mice (Roche - Cat. No. 1444581) in RPMI1640 (PAN - Cat. No. PO4-17500) at 37 °C and 5% CO ₂ .

Example 2

Inhibition of CSF-1 binding to CSF-1R (ELISA)

Assays were performed at RT in 384-well microtiter plates (MicroCoat, DE, catalog number 464718). After each incubation step, the plates were washed 3 times with PBST.

Initially, plates were coated with 0.5 mg/ml goat F(ab') ₂ biotinylated anti-Fcγ (Jackson ImmunoResearch., Cat. No. 109-006-170) for 1 hour.

-20和2%BSA的PBS (Roche DiagnosticsGmbH,DE)将孔封闭0.5小时。将75ng/ml huCSF 1R-huFc嵌合物（可溶性胞外域）固定化于板达1小时。然后，将纯化的抗体在PBS/0.05%Tween20/0.5%BSA中的稀释液温育1小时。在添加3ng/ml CSF-1(Biomol，DE，产品目录编号60530)、50ng/ml生物素化的抗CSF-1克隆BAF216(R&D Systems，UK)和1:5000稀释的链霉亲合素HRP(Roche Diagnostics GmbH，DE，产品目录编号11089153001)的混合物达1小时后，用PBST将板清洗6次。使用抗CSF-IRSC-02，克隆2-4A5(Santa Cruz Biotechnology,US)（其抑制配体-受体相互作用）作为阳性对照。用新鲜制备的BM bluePOD底物溶液（BM blue

3,3’-5,5’-四甲基联苯胺，Roche Diagnostics GmbH，DE，产品目录编号11484281001）将板于RT显现30分钟。于370nm测量吸光度。所有抗CSF-1R抗体都显示对CSF-1结合CSF-1R的显著抑制（见表1）。使用抗CSF-IR SC-02，克隆2-4A5(Santa Cruz Biotechnology,US)（其抑制配体-受体相互作用）作为参照对照。Thereafter, supplemented with 0.2%Tween

Wells were blocked with 20 and 2% BSA in PBS (Roche Diagnostics GmbH, DE) for 0.5 hours. 75ng/ml huCSF 1R-huFc chimera (soluble extracellular domain) was immobilized on the plate for 1 hour. Then, dilutions of purified antibodies in PBS/0.05% Tween20/0.5% BSA were incubated for 1 hour. After adding 3 ng/ml CSF-1 (Biomol, DE, catalog number 60530), 50 ng/ml biotinylated anti-CSF-1 clone BAF216 (R&D Systems, UK) and 1:5000 dilution of streptavidin HRP (Roche Diagnostics GmbH, DE, catalog number 11089153001 ) for 1 hour, the plate was washed 6 times with PBST. Anti-CSF-IRSC-02, clone 2-4A5 (Santa Cruz Biotechnology, US), which inhibits ligand-receptor interaction, was used as a positive control. With freshly prepared BM blue POD substrate solution (BM blue

3,3'-5,5'-Tetramethylbenzidine, Roche Diagnostics GmbH, DE, catalog number 11484281001) The plate was developed for 30 minutes at RT. Absorbance was measured at 370 nm. All anti-CSF-1R antibodies showed significant inhibition of CSF-1 binding to CSF-1R (see Table 1). Anti-CSF-IR SC-02, clone 2-4A5 (Santa Cruz Biotechnology, US), which inhibits ligand-receptor interactions, was used as a reference control.

Table 1: Calculated IC50 values for inhibition of CSF-1/CSF-1R interaction

Example 3

Inhibition of CSF-1-induced CSF-1R phosphorylation in NIH3T3-CSF-1R recombinant cells

4.5x10 ³ NIH 3T3 cells infected with the full-length CSF-1R expression vector with retrovirus in DMEM (PAA catalog number E15-011), 2mM L-glutamine (Sigma, catalog number G7513), 2 mM sodium pyruvate, 1x non-essential amino acids, 10% FKS (PAA, catalog number A15-649) and 100 μg/ml PenStrep (Sigma, catalog number P4333 [10 mg/ml]) until they reached confluency. Thereafter, the cells were treated with sodium selenite [5 ng/ml] (Sigma, catalog number S9133), transferrin [10 μg/ml] (Sigma, catalog number T8158), BSA [400 μg/ml] (Roche Diagnostics GmbH, catalog number 10735078), 4 mM L-glutamine (Sigma, catalog number G7513), 2 mM sodium pyruvate (Gibco, catalog number 11360), 1x non-essential amino acids (Gibco, catalog number: 11140- 035), 2-Mercaptoethanol [0,05 mM] (Merck, catalog number M7522), 100 μg/ml and PenStrep (Sigma, catalog number P4333) in serum-free DMEM medium (PAA catalog number E15-011) wash , and incubated in 30 μl of the same medium for 16 hours to allow receptor upregulation. 10 [mu]l of diluted anti-CSR-1R antibody was added to the cells for 1.5 hours. Cells were then stimulated with 10 μl of 100 ng/ml huM-CSF-1 (Biomol Cat# 60530) for 5 minutes. After incubation, the supernatant was removed, the cells were washed twice with 80 μl of ice-cold PBS, and 50 μl of freshly prepared ice-cold lysis buffer (150 mM NaCl/20 mM Tris pH 7.5/1 mM EDTA/1 mM EGTA/1 %Triton X-100/1 protease inhibitor tablet (Roche Diagnostics GmbH catalog number 1836170)/10 μl/ml Phosphatase inhibitor cocktail 1 (Sigma catalog number P-2850, 100x stock solution)/10 μl/ml protease inhibitor 1 (Sigma catalog number P-5726, 100x stock solution)/10 μl/ml 1M NaF). After 30 minutes on ice, the plate was shaken vigorously on a plate shaker for 3 minutes and then centrifuged at 2200 rpm for 10 minutes (Heraeus Megafuge 10).

溶液将板显现，并检测吸光度。数据以没有抗体情况中的阳性对照%计算，并且表示比率值磷酸/总受体。在没有添加M-CSF-1的情况中限定阴性对照。使用抗CSF-1R SC-02，克隆2-4A5（Santa Cruz Biotechnology，US，还可见Sherr,C.J.等,Cell 41(1985)665-676）（其抑制配体-受体相互作用）作为参照对照。Cell lysates were analyzed by Elisa for the presence of phosphorylated and total CSF-1 receptors. For detection of phosphorylated receptors, a kit from R&D Systems (Catalogue No. DYC3268-2) was used according to the supplier's instructions. For detection of total CSF-1R, 10 μl of lysate were immobilized on the plate by using the capture antibody contained in the kit. Thereafter, a 1:750 dilution of biotinylated anti-CSF-1R antibody BAF329 (R&D Systems) and a 1:1000 dilution of streptavidin-HRP conjugate were added. After 60 minutes, with freshly prepared ABTS

The solution visualizes the plate and the absorbance is measured. Data are calculated as % positive control in the absence of antibody and represent ratio values phospho/total receptor. A negative control was defined in the absence of M-CSF-1 addition. Anti-CSF-1R SC-02, clone 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, CJ et al., Cell 41 (1985) 665-676), which inhibits ligand-receptor interactions, was used as a reference control .

Table 2: Calculated IC50 values for inhibition of CSF-1 receptor phosphorylation

Example 4

Determination of the affinity of anti-CSF-1R antibodies for CSF-1R

Instrument: BIACORE A100

Chip: CM5 (Biacore BR-1006-68)

Coupling: amine coupling

Buffer: PBS (Biacore BR-1006-72), pH 7.4, 35°C

For affinity measurements, 36 μg/ml anti-mouse Fcγ antibody (from goat, Jackson ImmunoReasearch JIR115-005-071) was coupled to the chip surface to capture antibody against CSF-1R. CSF-1R ECD (R&D-Systems 329-MR or in-house subcloned pCMV-presS-HisAvitag-hCSF-1R-ECD) was added in solution at various concentrations. Binding was measured by injecting CSF-1R for 1.5 minutes at 35°C; dissociation was measured by washing the chip surface with buffer for 10 minutes at 35°C. Anti-CSF-IR SC-02, clone 2-4A5 (Santa Cruz Biotechnology, US; see also Sherr, C.J. et al., Cell 41 (1985) 665-676 ), which inhibits ligand-receptor interactions, was used as a reference control .

For the calculation of kinetic parameters, the Langmuir 1:1 model was used.

A100)测量的亲和力数据Table 3: At 35°C by SPR (BIACORE

A100) measured affinity data

Antibody K _D (nM) k _a (1/Ms) k _d (1/s) t _1/2 (minutes) <CSF-1R>7G5.3B6 0.61 8.0E+06 4.9E-03 2.37 SC-02, clone 2-4A5 2.73 5.09E+05 1.39E-03 8.31

Example 5

Epitope mapping of anti-CSF-1R monoclonal antibody based on cross-competition by utilizing SPR

A100Instrument: BIACORE

A100

Chip: CM5 (Biacore BR-1006-68)

Coupling: amine coupling

Buffer: PBS PBS (Biacore BR-1006-72), pH 7.4, 25°C

A100仪）的。For epitope mapping assays via cross-competition, 36 μg/ml anti-mouse Fcγ antibody or anti-rat Fcγ antibody (from goat, Jackson Immuno Research Cat. No. 115-005-071 and Cat. No. 112-005-071 ) coupled to the sensor chip surface to present antibodies against CSF-1R. After capture from 5 μg/ml anti-CSF-1R monoclonal antibody, block the free binding capacity of the capture antibody with 250 μg/ml mouse or rat immunoglobulin (Pierce Cat. No. 31202 and Pierce Cat. No. 31233), followed by injection 12.5 μg/ml CSF-1R (R&D-Systems catalog number 329-MR) for 2 minutes. Binding of the secondary anti-CSF-1R antibody was analyzed by injection for 2 minutes and dissociation was measured by washing with buffer for 5 minutes. Assays and measurements were performed at 25°C. Specific binding of the second anti-CSF-1R antibody was compared against spots with the same chip setup but only without CSF-1R injection. Cross-competition data have been calculated as a percentage (%) of the expected binding response of the second anti-CSF-1R antibody. The term "Percentage (%) of expected binding response" for secondary antibody binding is calculated by "100*relative response(mass_stability_early)/rMax", where rMax is calculated by "relative response(mass_stability_late)* Antibody Molecular Weight/Antigen Molecular Weight" calculation, as described in Biacore epitope localizer (for BIACORE

A100 meter).

Minimal binding responses were also calculated from pairs of the same antibody 1 and 2. The maximum value obtained by it + 100%, preferably 50% is set as the threshold of significant binding competition (see Table X, for example for antibody <CSF-1R> 7G5.3B6, the calculated threshold is 3+3=6, preferably 3 +1.5=4.5). Thus, an "anti-CSF-1R antibody that binds to the same epitope as <CSF-1R>7G5.3B6" has a percentage (%) of expected binding response of less than 6, preferably less than 4.5.

Anti-CSF-1R SC-02, clone 2-4A5 (Santa Cruz Biotechnology, US, see also Sherr, C.J. et al., Cell 41 (1985) 665-676 ), which inhibits ligand-receptor interactions, was used as a reference control .

Table 4: Epitope mapping of anti-CSF-1R antibodies via cross-competition data

The results indicate that the antibody that binds the same epitope as <CSF-1R>7G5.3B6 binds another epitope than SC-02, clone 2-4A5.

Example 6

Growth inhibition of NIH3T3-CSF-1R recombinant cells in 3D culture by anti-CSF-1R monoclonal antibody treatment (CellTiterGlo assay)

The NIH 3T3 cells infected with retrovirus will be supplemented with 2mML-glutamine with the expression vector of full-length wild type CSF-1R (SEQ ID No:15) or mutant CSF-1R L301S Y969F (SEQ ID No:16). Amide, 2 mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany) in DMEM high glucose medium (PAA, Pasching, Austria) in polyHEMA (poly(2-hydroxyethylmethyl) (Polysciences, Warrington, PA, USA)) (Polysciences, Warrington, PA, USA)) coated (to prevent adhesion to plastic surfaces). Cells were seeded in medium replaced with serum with 5 ng/ml sodium selenite, 10 mg/ml transferrin, 400 μg/ml BSA and 0.05 mM 2-mercaptoethanol. Upon treatment with 100 ng/ml huCSF-1 (Biomol, Hamburg, Germany), cells expressing wtCSF-1R formed dense spheroids that grew three-dimensionally, a property termed anchorage-independent. These spheroids closely resemble the three-dimensional structure and organization of solid tumors in situ. Mutant CSF-1R recombinant cells were able to form spheroids independently of CSF-1 ligand. Spheroid cultures were incubated for 3 days in the presence of 10 μg/ml antibody. CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells.

table 5:

^** Average of 15 different experiments,

^*** Average of 6 different experiments

Example 7

Growth Inhibition of BeWo Tumor Cells in 3D Culture by Anti-CSF-1R Monoclonal Antibody Treatment (CellTiterGlo Assay)

BeWo choriocarcinoma cells (ATCC CCL-98) were cultured in F12K medium (Sigma, Steinheim, Germany) supplemented with 10% FBS (Sigma) and 2 mM L-glutamine. ^5x104 cells/well were seeded in 96-well polyHEMA (poly(2-hydroxyethylmethacrylate)) coated plates containing F12K medium supplemented with 0.5% FBS and 5% BSA. Concomitantly, 200 ng/ml huCSF-1 and 10 μg/ml different anti-CSF-1R monoclonal antibodies were added and incubated for 6 days. CellTiterGlo assay was used to detect cell viability by measuring the ATP content of cells in relative light units (RLU). Inhibition of CSF-1 -induced growth was observed when BeWo spheroid cultures were treated with different anti-CSF-1R antibodies (10 μg/ml). To calculate antibody-mediated inhibition, the mean RLU value of unstimulated BeWo cells was subtracted from all samples. The mean RLU value of CSF-1 stimulated cells was arbitrarily set to 100%. Mean RLU values of cells stimulated with CSF-1 and treated with anti-CSF-1R antibody were calculated as % of CSF-1 stimulated RLU. Table 6 shows the calculated data; Figure 1 depicts the mean RLU values. Each mean value was derived from triplicate.

Table 6:

Example 8

Inhibition of Macrophage Differentiation/Monocyte Survival by Anti-CSF-1R Monoclonal Antibody Treatment (CellTiterGlo Assay)

Mononuclear cells were isolated from peripheral blood using RosetteSep ^™ Human Monocyte Enrichment Cocktail (StemCell Tech. - Cat. No. 15028). Incubate the enriched monocyte population at 37°C and 5% CO in _a solution supplemented with 10FCS (GIBCO - Cat. No. 011-090014M), 4 mM L-Glutamine (GIBCO - Cat. No. 25030) and 1xPenStrep (Roche 1 074440) in 100 μl RPMI 1640 (Gibco - Cat. No. 31870) into 96-well microtiter plates (2.5x104 cells/well). When 150 ng/ml huCSF-1 was added to the medium, clear differentiation into adherent macrophages could be observed. This differentiation can be inhibited by adding anti-CSF-1R antibody. Concomitantly, monocyte survival was affected and could be analyzed by CellTiterGlo (CTG) assay. IC50s were calculated from the concentration-dependent inhibition of monocyte survival by antibody treatment (see Table 7).

Table 7:

Antibody IC ₅₀ [nM] <CSF-1R>7G5.3B6 0.4 SC-02, clone 2-4A5 2.4

PCT

To be completed by the receiving Office

To be completed by the International Bureau

0-5 Date of receipt of this form by the International Bureau July 14, 2011 0-5-1 acceptance officer WAGNER,Nathalie

Form PCT/RO/134

Claims (16)

1. An antibody binding to human CSF-1R, characterized in that it binds to the same epitope as the deposited antibody DSM ACC2921. 2. The antibody according to claim 1, characterized in that the CDR3 region of SEQ ID NO: 1 is comprised as heavy chain variable domain CDR3 region. 3. Antibody according to claim 2, characterized in that: a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a). 4. The antibody according to claim 3, characterized in that it comprises a) the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 7, and the amino acid sequence of the light chain variable domain is SEQ ID NO: 8; or b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a). 5. Antibody according to any one of claims 1 to 4, characterized in that said antibody is of the human IgG4 subclass or of the human IgG1 subclass. 6. Pharmaceutical composition characterized in that it comprises an antibody or fragment according to any one of claims 1 to 5. 7. The antibody according to any one of claims 1 to 5 for use in the treatment of cancer. 8. The antibody according to any one of claims 1 to 5 for use in the treatment of bone loss. 9. The antibody according to any one of claims 1 to 5 for use in the prevention or treatment of metastasis. 10. An antibody according to any one of claims 1 to 5 for use in the treatment of an inflammatory disease. 11. Nucleic acid encoding the heavy chain of an antibody that binds CSF-1R, characterized in that said antibody comprises a variable domain according to claim 2, 3 or 4. 12. Expression vector characterized in that it comprises a nucleic acid according to claim 11 for expressing an antibody binding to CSF-1R in a prokaryotic or eukaryotic host cell. 13. A prokaryotic or eukaryotic host cell comprising a vector according to claim 12. 14. A method for producing a recombinant antibody according to any one of claims 1 to 5, characterized in that the nucleic acid according to claim 11 is expressed in a prokaryotic or eukaryotic host cell and obtained from said cell or cell culture The antibodies were recovered from the serum. 15. An antibody binding to human CSF-1R, characterized in that: a) The heavy chain variable domain comprises the CDR3 region of SEQ ID NO:1, the CDR2 region of SEQ ID NO:2, and the CDR1 region of SEQ ID NO:3, and the light chain variable domain comprises the CDR3 of SEQ ID NO:4 region, the CDR2 region of SEQ ID NO:5, and the CDR1 region of SEQ ID NO:6; or b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a). 16. The antibody according to claim 15, characterized in that it comprises: a) the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 7, and the amino acid sequence of the light chain variable domain is SEQ ID NO: 8; or b) A CDR-grafted, humanized or T-cell epitope-depleted antibody variant of the antibody of a).

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